Analysis of extended genomic rearrangements in oncological research.

Screening for genomic rearrangements is a fundamental task in the genetic diagnosis of many inherited disorders including cancer-predisposing syndromes. Several methods were developed for analysis of structural genomic abnormalities, some are targeted to the analysis of one or few specific loci, others are designed to scan the whole genome. Locus-specific methods are used when the candidate loci responsible for the specific pathological condition are known. Whole-genome methods are used to discover loci bearing structural abnormalities when the disease-associated locus is unknown. Three main approaches have been employed for the analysis of locus-specific structural changes. The first two are based on probe hybridization and include cytogenetics and DNA blotting. The third approach is based on PCR amplification and includes microsatellite or single nucleotide polymorphism (SNP) genotyping, relative allele quantitation, real-time quantitative PCR, long PCR and multiplex PCR-based methods such as multiplex ligation-dependent probe amplification and the recently developed nonfluorescent multiplex PCR coupled to high-performance liquid chromatography analysis. Whole-genome methods include cytogenetic methods, array-comparative genomic hybridization, SNP array and other sequence-based methods. The goal of the present review is to provide an overview of the main features and advantages and limitations of methods for the screening of structural genomic abnormalities relevant to oncological research.

Gastric adenomas: relationship between clinicopathological findings, Helicobacter pylori infection, APC mutations and COX-2 expression.

Gastric adenomas are rare neoplastic growths characterized by localized polypoid proliferations of dysplastic epithelium that tend to progress to infiltrating adenocarcinoma. Therefore, the identification of molecular markers that could reliably recognize adenomas at risk of progression is advocated in the clinical management. In this study we investigated, in a series of gastric adenoma specimens from an area at high risk of gastric cancer, the relationship between clinicopathological characteristics of adenoma and Helicobacter pylori infection, APC mutational status, and COX-2 and the down-stream enzyme mPGES1 expression. Helicobacter pylori infection, detected in 24%, and 33% by histology and PCR analyses, respectively, did not show any relationship with growth pattern, localization, size, dysplasia grade and presence of synchronous cancer. Pathogenetic mutations of MCR region (codons 1269-1589) of the APC gene were detected only in one case corresponding to a single, small size, low grade, H. pylori-negative adenoma. The expression of COX-2 largely matched that of mPGES(1). Both were overexpressed in 79% of cases showing a relationship with high-grade dysplasia, size >10 mm and presence of a synchronous carcinoma. In conclusion, COX-2 may play a key role in the development and progression of gastric adenoma and could be an attractive target in the management of gastric adenoma at major risk of cancer development.

Thyroid carcinoma usually occurs in patients with familial adenomatous polyposis in the absence of biallelic inactivation of the adenomatous polyposis coli gene.

Papillary thyroid carcinoma (PTC) is a rare extracolonic manifestation of familial adenomatous polyposis, determined by germline mutations of the adenomatous polyposis coli (APC) gene. The aim of this study was to assess the presence of loss of heterozygosity of APC in the thyroid tumoral tissue. Specimens from six female patients, aged 20-36, were analyzed for germline and somatic mutations of the APC gene by restriction enzyme analysis and sequence analysis. Five of the six also had analysis for ret/PTC, a chimeric gene, the activation of which is restricted to papillary TC. Because a previous study showed that germline mutations in familial adenomatous polyposis-associated thyroid carcinoma were located between codons 140 and 1513, the search for somatic mutations of the APC gene was restricted to this genomic area. Three of the six patients, belonging to the same kindred, had a germline mutation at codon 1061. The remaining three, one per kindred, had germline mutations at codons 1061, 1061, and 1309, respectively. None of the six patients had loss of heterozygosity for APC or somatic mutation in the explored genomic area (codon 545 and codons 1061-1678). Four of five had activation of ret/PTC in the thyroid tumoral tissue, as ret/PTC1 isoform. Either APC has a tissue-specific dominant effect in the thyroid gland or the germline mutation confers a generic susceptibility to cancer development, but other factors (sex-related factors, environmental radiation, modifier genes) are also required for TC development. This usually involves ret/PTC activation, suggesting a possible cooperation between altered function of APC and gain of function of ret.

Germline mutations of the APC gene in patients with familial adenomatous polyposis-associated thyroid carcinoma: results from a European cooperative study.

Papillary thyroid carcinoma (PTC) is one extracolonic manifestation affecting about 1-2% of patients with familial adenomatous polyposis (FAP). Ninety-seven patients with FAP-associated PTC have previously been reported, including 6 pairs of siblings. During a European collaborative study, 15 patients with FAP-associated PTC were collected. All 15 patients were females. The mean age at thyroidectomy was 24.9 yr (range, 19-39 yr). In 13 subjects, APC germline mutations had been detected; they were at codons 140, 593, 778, 976, 993, 1061 (n = 5), 1105 (n = 1), and 1309 (n = 2), respectively. A review of the literature added 11 other patients with FAP-associated PTC and detection of germline APC mutations; they were at codons 313 (n = 2), 698 (n = 3), 848 (n = 2), 1209 (n = 2), 1061 (n = 1), and 1105 (n = 1), respectively. The latter led to formation of the same stop codon (TAA) at 1125-1126 as the mutation at codon 1061. Therefore, 21 of 24 mutations were in exon 15 in the genomic area usually associated with congenital hypertrophy of the retinal pigment epithelium (CHRPE), i.e. codons 463-1387. Typical CHRPE was found in 17 of 18 affected patients who had specific screening. Interestingly, 22 of the 24 patients had their mutation out of the mutation cluster region (codons 1286-1513), which is currently considered the hot spot mutation area, in particular for extracolonic manifestations of FAP. The difference in the incidence of germline mutations before and after codon 1220 between PTC and non-PTC FAP patients was statistically significant (P<0.05) for both patients and kindreds (P = 0.005 and P = 0.049, respectively). Even if most mutations were scattered throughout the entire 5'-portion of exon 15, 8 of 23 patients (6 with mutation at 1061 and 2 with mutation at 1105; i.e. more than one third) had the same truncated protein product. The awareness that patients with PTC usually have APC mutations that cluster in a well defined genomic area, in addition to giving a deeper insight into gene function, could facilitate both earlier diagnosis and better treatment. In particular, intensive screening for thyroid nodules after age 15 yr is recommended when a single patient or an entire kindred have CHRPE and/or mutations in the 5'-portion of exon 15.

APC gene mutations and colorectal adenomatosis in familial adenomatous polyposis.

Correlations between germline APC mutation sites and colorectal pathophenotypes, as evaluated by the direct count of adenomas at colectomy, were investigated analysing colectomy specimens from 29 FAP patients carrying one mis-sense (codon 208) and 14 frame-shift or non-sense APC mutations (codons 232, 367, 437, 623, 876, 995, 1061, 1068, 1075, 1112, 1114, 1309, 1324, 1556). The mis-sense mutation at codon 208 was associated with a relatively mild colorectal pathophenotype. The mutation at codon 367, subject to alternative splicing, was associated with attenuated FAP. The mutation at codon 1309 was associated with the profuse colorectal adenomatosis. For 13 mutations, predicted to result in null alleles or truncated APC proteins, we correlated density and distribution of colorectal adenomas with the predicted functional effects of the mutation. The most severe colorectal pathophenotype was significantly associated with the truncating mutation at codon 1309, which is located downstream to the I beta-catenin binding domain but upstream II beta-catenin-binding domain. Mutations between codons 867 and 1114, which affect the I beta-catenin binding domain, as well as mutations occurring in exons 6 and 9, predicted to result in null alleles, were associated with a less severe colorectal pathophenotype. Overall, the highest number of adenomas was detected in the right colon, followed by the left colon, transverse colon sigma and rectum. However, the highest density of adenomas was observed in the left colon, followed by the right colon, sigma, transverse colon and rectum. Colorectal carcinomas, observed in only five patients, were all in the left colon.

Unbalanced germ-line expression of hMLH1 and hMSH2 alleles in hereditary nonpolyposis colorectal cancer.

We analyzed the hMLH1 and hMSH2 genes in 30 unrelated hereditary nonpolyposis colorectal cancer (HNPCC) patients using mutational and immunohistochemical analyses combined whenever possible with primer extension assays, designed to estimate hMLH1 and hMSH2 transcript expression in peripheral blood lymphocytes. Single-strand conformational polymorphism screening and PCR-direct sequencing revealed seven hMLH1 and five hMSH2 sequence variants in 14 unrelated HNPCC patients, including three definite pathogenic mutations, four amino acid substitutions of uncertain pathogenic significance, and five polymorphisms. Immunohistochemistry indicated the lack of either hMLH1 or hMSH2 protein expression in tumors from 13 patients, and the absence of both hMLH1 and hMSH2 immunostaining was observed in the tumor from one additional case. The lack of hMLH1 or hMSH2 immunostaining was associated with the presence of microsatellite instability in the corresponding tumor and was also observed in tumors from patients negative for pathogenic mutations by mutational screening. There was a marked unbalance in the allelic expression of either hMLH1 or hMSH2 transcripts in three of eight unrelated HNPCC patients that could be analyzed, although a less marked unbalance was detected in two additional patients. Tumors from patients with germ-line unbalance in hMLH1 or hMSH2 transcript expression did not express the corresponding mismatch repair protein and displayed microsatellite instability. Our results indicate that constitutional alterations in hMLH1 and hMSH2 transcript expression may represent genetic markers for HNPCC carrier status also in cases in which mutational analysis did not detect a definite pathogenic variant. This suggests that transcript deregulation may represent a relevant mode of germ-line inactivation for mismatch repair genes.

Transcripts with splicings of exons 15 and 16 of the hMLH1 gene in normal lymphocytes: implications in RNA-based mutation screening of hereditary non-polyposis colorectal cancer.

Germline mutations of the hMLH1 gene are estimated to account for a large fraction of kindreds affected by hereditary non-polyposis colorectal cancer (HNPCC). In a significant number of cases, hMLH1 mutations result in the expression of truncated proteins. We report here two novel alternatively spliced forms of hMLH1 mRNA in normal lymphocytes. One of these novel isoforms lacks the coding region of the gene between codons 557 and 578, corresponding to the entire exon 15. The deletion introduces a frameshift that results in a premature stop signal. The other isoform is characterised by an in-frame deletion spanning codons 578-632, corresponding to loss of the entire exon 16. Further studies are necessary to establish the biological significance of these alternative splicings. The presence of alternatively spliced hMLH1 transcripts that mimic pathogenic mutations should be taken into account in the mutational screening of the hMLH1 gene by reverse transcription-polymerase chain reaction methodologies.

Optimized PCR labeling in mutational and microsatellite analysis.

To optimize the labeling and visualization of PCR products we tested different variables, including deoxynucleotide concentration and ratio, dilution of labeled product, number of PCR cycles, and use of one-step or nested labeling protocols. Labeling was achieved using a fixed amount of labeled dATP, whose relative specific activity was varied by adding increasing amounts of cold dATP. Optimal PCR-labeling intensity was reached at dATP concentrations between 0.9 and 7.0 micromol/L, with a peak at 1.8 micromol/L. This concentration corresponded to an optimal ratio between the increase in specific activity and the decrease in DNA yield. Nucleotide imbalances >1:2 were not advantageous. Mutational analysis by single-strand conformational polymorphism (SSCP) was used to validate PCR-labeling protocols. The limiting nucleotide concentrations did not affect SSCP. Clear SSCP patterns were obtained using DNA templates of different sizes derived from several genes. SSCP patterns obtained using one-step or nested PCR-labeling protocols were equivalent and were visualized after overnight exposure, using [alpha35S]dATP as the label. Dilutions of labeled products ranging between 1:10 and 1:2.5 influenced SSCP patterns, and the lowest dilution tested produced better-defined and more-intense signals. Optimized SSCP conditions allowed the detection of novel and previously characterized nucleotide variants. Clear microsatellite typing was also obtained using optimized protocols and [alpha35S]dATP as the label.

Transcript dosage effect in familial adenomatous polyposis: model offered by two kindreds with exon 9 APC gene mutations.

Analysis of genotype-phenotype correlations in familial adenomatous polyposis (FAP) patients demonstrated that the phenotypic heterogeneity of FAP is partly related to the mutation site. We investigated the molecular basis for the difference in severity of colorectal disease observed comparing FAP patients from two kindreds with neighbouring germline mutations in exon 9 of the APC gene. Patients from one kindred presented with a attenuated form of FAP, characterized by a low number of colorectal adenomas (up to 22). In FAP patients from this kindred, the APC gene mutation was localized at codon 367, in the portion of exon 9 that is alternately spliced. This is expected to result in the splicing-out of the mutation site in a fraction of mRNA molecules and in the residual production of wild-type transcripts from the mutant APC allele. Patients from the other kindred manifested a FAP phenotype characterized by hundreds of colorectal adenomas (320 to > 500). In these patients, the APC gene mutation abolished the donor site of exon 9a, used in both alternately spliced isoforms of the exon. The analysis of the relative levels of mutant and wild-type transcripts in unaffected colonic mucosa demonstrated that the mutant allele was not expressed. The model offered by our FAP patients with neighbouring exon 9 APC mutations supports the view that in addition to the mutation site, the type of mutation and transcript dosage effects contribute to the heterogeneity of disease phenotypes.

Congenital hypertrophy of the retinal pigment epithelium in familial adenomatous polyposis. Novel criteria of assessment and correlations with constitutional adenomatous polyposis coli gene mutations.

BACKGROUND: Congenital hypertrophy of the retinal pigment epithelium (CHRPE) is the most common extracolonic manifestation of familial adenomatous polyposis (FAP) and is an early clinical marker of the disease. It seems to be correlated with the position of constitutional mutations of the adenomatous polyposis coli (APC) gene. METHODS: The authors investigated the expression of CHRPE and its correlation with the position of the APC gene in FAP patients and in "at risk" relatives from 31 FAP kindreds. To obtain comparable data on CHRPE expression, the authors developed a novel scoring system based on morphologic and dimensional criteria. RESULTS: A positive CHRPE score was obtained in 29 of 39 FAP patients (74%) and in 16 of 53 relatives who showed no clinical evidence of FAP (30%). Colonoscopy revealed polyps in 20 of the 47 relatives who could be examined. The cumulative sensitivity and specificity of CHRPE were 72.88% and 96.29%, respectively. APC gene mutations were characterized in 34 subjects from 17 kindreds. In 28 of the subjects, mutations were detected in exon 15, between codons 876 and 1324. Mutations were found in exon 9 in 6 subjects. In 3 of the 6 subjects, they were found at the site where both forms of alternative splicing of the exon occur (codon 437). In the other 3 subjects (another kindred), mutations were found in the portion of exon 9 in which alternative splicing occurs (codon 367). Only 1 of the 6 subjects (16.6%) with mutations in exon 9 had a positive CHRPE score, compared with 28 of 28 subjects (100%) with mutations in exon 15. None of the 3 subjects with mutations in codon 437 had a positive CHRPE score. The CHRPE scores of exon 15 mutation carriers varied markedly both within and among kindreds, irrespective of the mutation site. CONCLUSIONS: The results of this study indicate that the site of APC gene mutation influences CHRPE expression but is not the only factor responsible for the presence and level of retinal lesions in FAP patients.

Novel allele of the hMLH1 gene bearing a TTC deletion in the 3' untranslated region.

SSCP analysis of the hMLH1 gene in two kindreds affected by hereditary nonpolyposis colorectal cancer (HNPCC) revealed the presence of unique conformers in all patients affected by colorectal cancer. Sequence analysis of the corresponding region of the gene revealed a 3 base pairs deletion within a TTC tandem repeat (G TTC TCC T-->G TTC T) beginning 29 base pairs downstream of the termination codon of the gene in the 3' untranslated region. This deletion causes the loss of an MboII restriction site. Analysis extended to 113 healthy unrelated individuals and 27 unrelated HNPCC patients demonstrated the occurrence of this novel variant of the hMLH1 gene at similar frequencies in unrelated HNPCC patients (3.7%) and in control individuals (2.2%). The allele bearing the TTC deletion appears to be expressed at levels comparable to those of the wild-type allele.

Cooperative signaling of ErbB3 and ErbB2 in neoplastic transformation and human mammary carcinomas.

In the present study we demonstrate that erbB-3 and erbB-2 cooperate in neoplastic transformation. Under conditions in which neither gene alone induced transformation, they readily transformed NIH3T3 cells if co-expressed. Furthermore, at high expression levels of ErbB2 which cause transformation, ErbB3 enhanced focus formation by one order of magnitude. Synergy required an intact ErbB2 extracellular domain and tyrosine kinase activity. Cooperation between ErbB3 and ErbB2 involved heterodimerization and increased tyrosine phosphorylation of ErbB3. Signaling by the heterodimer resulted in increased PI 3-kinase recruitment as well as quantitative and qualitative differences in substrate phosphorylation. Evidence for signaling by an active ErbB3-ErbB2 heterodimer in four mammary tumor cell lines indicated relevance of this mechanism for human neoplasia. Our detection of the NDF/heregulin transcript in NIH3T3 cells implicates an autocrine loop involving this ligand in signaling by the ErbB3-ErbB2 heterodimer in the model system, whereas heregulin-independent mechanisms likely exist for cooperative signaling by ErbB3 and ErbB2 chronically activated in some human mammary carcinomas.

Multiplex PCR analysis and genotype-phenotype correlations of frequent APC mutations.

Germline mutations of the adenomatous polyposis coli (APC) gene tend to cluster in discrete regions. Some of these mutations occur frequently in familial adenomatous polyposis coli (FAP) patients, and strategies for genetic diagnosis of the disease should include simple methods for their detection. We studied a total of 48 FAP-affected or "at-risk" members from 31 unrelated FAP pedigrees. Unrelated patients were analyzed using heteroduplex analysis on agarose minigels (HAAM) and multiplex allele-specific PCR. This novel strategy readily and reliably detected the three frequently occurring APC deletions at codons 1061, 1068, and 1309, allowing identification of mutant alleles in nine unrelated patients. A targeted mutational analysis, based on HAAM and amplification refractory mutation system (ARMS), allowed the rapid identification of 11 additional subjects with germline deletions, among relatives of the patients in whom mutations had been detected by multiplex PCR and HAAM. The use of two independent PCR-based tests, employing distinct sets of primers, reduces the possibility that artifacts occurring during DNA amplification may interfere with the diagnostic evaluation. The analysis of genotype-phenotype correlations provided evidence for heterogeneity with regard to the extent of colonic and extracolonic manifestations of the disease in subjects bearing identical mutations. However, the consistent association of the deletion at codon 1309 with more severe colonic disease than that observed in patients with mutations at codons 1061 and 1068, supports a correlation between mutation site and penetrance of FAP.

HTLV-1-associated myeloneuropathy in an Italian.

A 58-year old man presented with slowly progressive spastic paraparesis, ataxia, absent ankle jerks, bladder disturbances, impairment of vibration sense and mental deterioration. Electrophysiological studies documented axonal sensory neuropathy, posterior column and optic nerve involvement. Serum tests for anti-HTLV-1 antibodies were negative but HTLV-1 proviral sequences were consistently demonstrated in white blood cell genomic DNA using the polymerase chain reaction technique. Western blot and polymerase chain reaction assays of sera and DNA from family members were negative for HTLV-1. The most likely cause of infection in this patient was a blood transfusion received 2 years before onset of symptoms. This is the second Italian case of HTLV-1 associated myelopathy and the fourth reported in white subjects living in Europe.